A common and central regulatory mechanism in the cell is proline-directed phosphorylation on certain Ser/Thr- Pro (phos.Ser/Thr-Pro) motifs. The unique stereochemistry of Pro means that it can adopt two distinct cis or trans conformations, but the biological significance of these conformational switches was unappreciated for a long time. We have recently identified a unique prolyl isomerase, Pin1 (Gene ID, NM_006221; Protein ID, NP_006212) that catalyzes the conformational switches of specific phos.Ser/Thr-Pro motifs in a subset of proteins to regulate cell signaling. Such Pin1-catalyzed conformational regulation can have a profound impact on many key proteins in diverse cellular processes. Importantly, Pin1 deregulation plays a pivotal role in the development of an increasing number of diseases and provides a potential new therapeutic option. However, chemical probes to inhibit Pin1 function identified so far either lack the critically needed specificity or simply cannot enter cells. We have now developed a series of robust and sensitive procedures to identify and evaluate Pin1 probes in vitro and in vivo and also identified interesting hits in our pilot screen. Therefore, in this proposal, we will collaborate with Drs. Douglas Auld and Craig Thomas at NIH Chemical Genomics Center to identify Pin1 chemical probes, with the following specific aims: (1) To identify inhibitors of human Pin1 using a quantitative high-throughput screening approach against the MLSMR containing 300,000 small molecules. (2) To validate the potency and specificity of the hits in secondary assays to identify those compounds that specifically inhibit Pin1, but not other non-phosphorylation-specific prolyl isomerases. (3) To characterize and optimize Pin1 chemical probes by tertiary cell-based assays and structure-activity relationship analysis, structure-based methods, analogue synthesis and medicinal chemical principles. (4) Beyond the granting period for this proposal, to test the most promising compounds for their ability to inhibit Pin1 function in several Pin1-relevant mouse models of cancer or Alzheimer<s disease that we have established in our laboratory. This proposal would allow us to identify urgently needed chemical probes to study Pin1-regulated Pro-directed phosphorylation signaling under physiological and pathological conditions. PUBLIC HEALTH RELEVANCE: We have recently identified a new enzyme called Pin1 that is a pivotal regulator of numerous cellular processes. Importantly, Pin1 deregulation plays a critical role in the development of an increasing number of human diseases, including aging, cancer and Alzheimer<s disease. However, currently there is no Pin1-specific chemical probe available. In this application, we propose to discover and optimize Pin1 chemical probe to study Pin1-regulated processes under physiological and pathological conditions.